Our long-term goal is to understand how host factors function in human cells at the interface of maintaining nucleotide homeostasis and initiating viral defense mechanisms. The battle between viral pathogens like HIV and the primate immune system has placed nucleic acid metabolism center stage. For example, the restriction factors SAMHD1 and APOBEC3D/F/G/H dominantly interfere with HIV replication and are counteracted by viral Vpx and Vif proteins, respectively. Mutations in the SAMHD1 gene cause the autoimmune disease Aicardi-Goutieres syndrome (AGS), a lupus-like neurodegenerative disorder that clinically mimics congenital viral infection. Key insights into the interferon-mediatd innate immune response to nucleic acids have been gleaned from the genetics of AGS. AGS is caused by variations in the nucleic acid metabolizing enzymes SAMHD1, TREX1, the three-subunit RNase H2 complex, and ADAR1. Our work on the biochemistry and structure of SAMHD1 revealed the deoxynucleotide triphosphohydrolase activity and suggested a mechanism by which this enzyme might function at the interface of nucleic acid metabolism and the interferon-mediated antiviral response, serving normally to regulate cellular dNTP levels and during an interferon response to starve HIV of dNTPs required for reverse transcription. However, the details of the mechanism of SAMHD1 action in human cells and in HIV pathogenesis during immune activation are not well understood. In particular, the mechanisms by which this enzyme is regulated and how dysfunctional variants of SAMHD1 trigger nucleic acid-mediated innate immune responses and autoimmune disease have not been defined. In this project we will perform biochemical, genetic, and structural studies to generate a comprehensive understanding of SAMHD1 and its roles in nucleic acid metabolism, antiviral defense, and autoimmunity. Insights into these mechanisms will uncover new opportunities for therapeutic targeting in the antiviral response, autoimmunity, and inflammation.